Patent Application
20210230710
The present invention relates to methods for detecting an antiviral drug resistant Herpes Simplex Virus (HSV), and for diagnosing an infection with an antiviral drug-resistant HSV comprising the use of an HSV mutation/polymorphism.
Herpes simplex is a viral disease caused by the herpes simplex virus and is categorised based on the part of the body infected. Oral herpes involves the face or mouth. It may result in small blisters in groups often called cold sores or fever blisters or may just cause a sore throat. Genital herpes may form blisters that break open and result in small ulcers. Infection with HSV results in significant discomfort and may result in fever, muscle pains, swollen lymph nodes and headaches.
HSV comprises type 1 (HSV-1) and type 2 (HSV-2). HSV-1 more commonly causes infections around the mouth while HSV-2 more commonly causes genital infections. They can be transmitted by direct contact with body fluids or lesions of an infected individual and transmission may still occur when symptoms are not present. Genital herpes is classified as a sexually transmitted infection. It may be spread to an infant during childbirth. After infection, the viruses are transported along sensory nerves to the nerve cell bodies, where they reside lifelong. Recurrence of the viral infection can occur during immune suppression; for example, in patients undergoing bone marrow or solid organ transplantation, anticancer therapy or individuals in high-risk groups, for example, pregnant women, newborns and the elderly.
Over 70% of the adult population in the UK is infected with HSV. The infection is of clinical significance in immunocompromised individuals especially those undergoing bone marrow transplantation (BMT) and is treated with antivirals such as acyclovir (ACV) and penciclovir (PCV) (first line drugs), as well as foscarnet and cidofovir (second-line drugs). However, HSV (e.g. HSV-1 and HSV-2) can develop resistance to one of more of these drugs, and there is evidence that resistance to these treatments is increasing.
ACV and PCV are guanine analogues that act as chain terminators and block viral replication by inhibiting the viral DNA polymerase. Both drugs need to be triphosphorylated to become active and the first phosphorylation step is carried out by the HSV thymidine kinase (TK). Resistance to the drugs develops usually via the development of mutations (e.g. non-synonymous mutations) in the TK genes (UL23) and occasionally in the viral DNA polymerase genes (UL30). The resistance mutations are variable and consist of substitutions, deletions and insertions. The latter two types of mutations often occur in homopolymeric regions (usually a run of Cs or Gs) and often result in premature stop codons. Substitutions often result in corresponding amino acid sequence substitution polymorphisms. The antivirals foscarnet (FOS) and cidofovir (CDV) are viral DNA polymerase inhibitors (that are often used as second-line therapy) do not require activation by viral TK. Therefore, resistance to these two drugs usually develops via emergence of mutations in viral DNA polymerase and can result in cross-resistance to ACV and PCV.
Currently, there is no reliable test for drug-resistant HSV based on sequence analysis (e.g. detecting mutations/polymorphisms). Diagnosis of an antiviral drug-resistant HSV infection relies on assays based on assessing the phenotype of a virus. The gold standard assay involves phenotypic drug susceptibility testing using a plaque reduction assay. This requires isolation of virus (e.g. whole intact virus) which is difficult and time consuming. Therefore, the assay has long turnaround times of weeks; however, it is well standardised. Nonetheless, due to the cost and expertise required it is usually only performed by reference laboratories.
The interpretation of the potential resistance-causing effect of any mutation/polymorphism by genotypic methods is complex. Substitution mutations/polymorphisms are particularly difficult to interpret as the number of ‘natural’ mutations/polymorphisms in HSV, which do not result in drug resistance, vastly outnumbers the number of resistance-causing mutations/polymorphisms (e.g. substitutions), such that it is very difficult to determine resistance based on the sequence of TK or DNA pol alone. Therefore, it is highly unexpected that any one mutation (e.g. substitution) will cause resistance. This problem is exacerbated due to the fact that a public database of known and phenotypically characterised drug resistance-associated mutations/polymorphisms does not exist. While a number of resistance-associated mutations have previously been reported, these mutations were not interpreted/verified via a standardised assay and thus the resistance associated with these mutations is not directly comparable.
The present invention solves at least one of the above-mentioned problems.
In one aspect there is provided a method for detecting an antiviral drug-resistant HSV (e.g. in a subject), comprising:
The term “indicative” as used in the context of “the absence of said one or more HSV mutation is indicative of the absence of an antiviral drug-resistant HSV” means that the HSV may be less likely to be an antiviral drug-resistant HSV when compared with an HSV comprising said mutation. The skilled person will understand that antiviral drug-resistance may (regardless) be conferred by an alternative mutation.
Mutations followed by “HSV-1” in parentheses may be identified in a HSV-1 virus. Mutations followed by “HSV-2” in parentheses may be identified in a HSV-2 virus.
Surprisingly, these mutations find utility as robust markers for detecting antiviral drug-resistant HSV. Furthermore, a comprehensive database of such mutations (including associated HSV phenotypes) is provided together with algorithms for interpreting the phenotype resulting from a given mutation or mutation profile.
For the first time, the present inventors have genotyped numerous clinical isolates comprising antiviral drug-resistant HSV with a standardised assay, and have generated an extensive list of mutations which are particularly suitable for use in detecting an antiviral drug-resistant HSV and for diagnosis with an infection of the same. Advantageously, each of these mutations (as well as previously identified mutations) have been verified by the gold standard plaque reduction assay, such that the resulting phenotypes are directly comparable.
The same assay was also used to validate the phenotype of a large number of mutations not associated with antiviral drug-resistance (e.g. natural) mutations/polymorphisms.
In a preferable embodiment, said thymidine kinase (TK) mutation is selected from 250G>A (HSV-2), 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1). In a preferable embodiment, said DNA polymerase (DNA pol) mutation is selected from 1882C>G (HSV-2), 2405T>G (HSV-1), 2500G>T (HSV-1), 2515A>G (HSV-1), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and 2895_2896insT (HSV-1).
In a preferable embodiment, said TK polymorphism selected from E84K (HSV-2), Q34* (HSV-1), Q90* (HSV-2), Q125* (HSV-1), L49R (HSV-1), M1211 (HSV-1), 1166N (HSV-1), Q186H (HSV-2), H214R (HSV-2), E146fs (HSV-1), D228* (HSV-1), Y239H (HSV-1), L313S (HSV-2), T183* (HSV-1), H58fs (HSV-1), M85* (HSV-1), P154fs (HSV-2), M183* (HSV-2), A294fs (HSV-1), P295fs (HSV-1), and E296fs (HSV-1). In a preferable embodiment, R628G (HSV-2), L802R (HSV-1), A834S (HSV-1), T839A (HSV-1), F965_I966insF (HSV-1), and 1966* (HSV-1).
In one embodiment, the mutation is a pyridine to pyridine substitution. In one embodiment, the mutation is a pyrimidine to pyrimidine substitution.
The mutation/polymorphism nomenclature used herein is described for example in J. T. den Dunnen, S. E. Antonarakis: Hum Genet 109(1): 121-124 (2001), which is incorporated herein by reference. Mutations may be detected in genomic DNA, cDNA or RNA. Optionally, a description of a mutation may be preceded by a sign selected from g., c., and r to denote identification of a mutation in DNA, cDNA or RNA, respectively. For example, a mutation 748G>C in genomic DNA may be referred to as g.748G>C.
This method may be performed on a sequence (e.g. nucleic acid sequence) comprised within a HSV sequence (e.g. comprised within a TK and/or DNA pol sequence). Said sequence may be determined (e.g. obtained) either prior to carrying out the present method or at the same time as carrying out the present method. In one embodiment, the method comprises identifying the presence or absence of said one or more HSV mutation in a sequence, preferably a TK and/or DNA pol sequence. In one embodiment the presence or absence of said one or more HSV mutation is detected in a database of HSV mutations, wherein said database comprises said one or more HSV mutation.
The methods of the present invention may be performed in silico e.g. by aligning a HSV sequence with the sequence of a reference HSV (e.g. wild-type HSV). Alternatively, the sequence may be investigated (e.g. parsed) for the presence of one or more of said HSV mutations.
In a further aspect, the invention provides a method for treating an infection of an antiviral drug-resistant HSV in a subject, comprising:
In a preferable embodiment, said thymidine kinase (TK) mutation is selected from 250G>A (HSV-2), 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1).
Suitably, step a. may further comprise confirming said antiviral drug-resistant HSV does not comprise a DNA Pol mutation associated with resistance to an antiviral drug. In one embodiment, step a. further comprises confirming said antiviral drug-resistant HSV does not comprise a DNA Pol mutation is selected from 1879C>G (HSV-1), 1882C>G (HSV-2), 2405T>G (HSV-1), 2420T>G (HSV-2), 2500G>T (HSV-1), 2515G>T (HSV-2), 2515A>G (HSV-1), 2530A>G (HSV-2), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), 2895_2896 insT (HSV-1), 2907_2908 insT (HSV-2), 2908_2909 insT (HSV-2), 2909_2910 insT (HSV-2), and 2910_2911 insT (HSV-2) (preferably 18820>G (HSV-2), 2405T>G (HSV-1), 2500G>T (HSV-1), 2515A>G (HSV-1), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and/or 2895_2896 insT (HSV-1)).
In one embodiment, step b. comprises administering to the subject a non-acyclovir and/or non-penciclovir drug. In one embodiment, step b. comprises administering to said subject a drug selected from a foscarnet, a cidofovir drug, a docosanol drug, BAY 54-6322, ASP2151 and BAY 57-1293.
In another aspect, there is provided a method for treating an infection of an antiviral drug-resistant HSV in a subject, comprising:
In a preferable embodiment, said DNA polymerase (DNA pol) mutation is selected from 18820>G (HSV-2), 2405T>G (HSV-1), 2500G>T (HSV-1), 2515A>G (HSV-1), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and 2895_2896 insT (HSV-1).
Suitably, step a. may further comprise confirming said antiviral drug-resistant HSV does not comprise a TK mutation associated with resistance to an antiviral drug. In one embodiment, step a. further comprises confirming said antiviral drug-resistant HSV does not comprise a TK mutation selected from 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 3760>T (HSV-2), 146T>G (HSV-1), 250G>A (HSV-2), 253A>C (HSV-1), 256A>C (HSV-2), 363G>A (HSV-1), 366G>A (HSV-2), 497T>A (HSV-1), 500T>A (HSV-2), 558G>T (HSV-2), 715T>C (HSV-1), 718T>C (HSV-2), 935T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 276delG (HSV-2), 278delG (HSV-2), 279delG (HSV-2), 280delG (HSV-2), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1) (preferably 250G>A (HSV-2), 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and/or 885delC (HSV-1)).
In one embodiment, step b. above comprises administering to the subject a non-acyclovir drug, a non-penciclovir drug, a non-foscarnet drug and/or a non-cidofovir drug.
The methods of the invention are intended to encompass all known treatments for an HSV infection.
In one embodiment, the method comprises confirming that an antiviral drug-resistant HSV comprises a DNA Pol mutation selected from 2405T>G (HSV-1), 2420T>G (HSV-2), 2500G>T (HSV-1), and 2515G>T (HSV-2) (preferably 2405T>G (HSV-1), and/or 2500G>T (HSV-1); and administering to said subject a cidofovir drug.
In one embodiment, the method comprises confirming that an antiviral drug-resistant HSV comprises a DNA Pol mutation selected from 2515A>G (HSV-1), and 2530A>G (HSV-2) (preferably 2515A>G (HSV-1)); and administering to said subject a foscarnet and/or cidofovir drug.
In one embodiment, the method comprises confirming that an antiviral drug-resistant HSV comprises a DNA Pol mutation selected from 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), 2895_2896 insT (HSV-1), 2907_2908 insT (HSV-2), 2908_2909 insT (HSV-2), 2909_2910 insT (HSV-2), and 2910_2911 insT (HSV-2) (preferably from 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), 2895_2896 insT (HSV-1)); and administering to said subject an acyclovir drug, a penciclovir drug, and/or a foscarnet drug.
In one embodiment, the method comprises confirming that an antiviral drug-resistant HSV comprises a DNA Pol mutation selected from 18790>G (HSV-1), and 18820>G (HSV-2) (preferably 18820>G (HSV-2)); and administering to said subject a cidofovir drug.
In one embodiment, the method comprises confirming that an antiviral drug-resistant HSV comprises a TK and/or DNA pol mutation as described herein (e.g. a TK and/or DNA pol mutation associated with resistance to an antiviral drug) and administering to a subject a drug selected from a docosanol drug, BAY 54-6322, ASP2151 and BAY 57-1293.
The absence of one or more HSV mutation as described herein is indicative of the absence of an antiviral drug-resistant HSV. Various further resistance-associated mutations exist, such that there may be presence of an antiviral drug-resistant HSV when the absence of one or more HSV mutation described herein is identified. As such, the absence of one or more of said HSV mutation is an indicator of the absence of an antiviral drug-resistant HSV.
In one embodiment, a method of the invention comprises identifying (or confirming the presence of) one or more further HSV TK and/or HSV mutation.
In one embodiment, a method of the invention comprises identifying one or more further HSV-1 TK mutation selected from 11_12 insC, 12_13 insC, 13_14 insC, 14_15 insC, 15_16 insC, 122G>A, 1510>T, 158A>G, 157T>G, 157T>C, 159T>A, 163G>A, 166G>T, 177G>C, 170C>A, 172C>A, 173A>G, 175G>C, 175G>T, 186A>C, 187A>G, 1880>T, 194C>A, 1990>T, 2210>G, 222C>A, 238T>A, 247G>T, 2520>T, 2500>T, 184delA, 185delA, 186delA, 187delA, 180delG, 181delG, 182delG, 183delG, 259T>C, 262T>C, 2650>T, 283G>A, 307A>C, 3100>T, 312A>T, 3100>T, 314A>C, 181_182 insG, 182_183insG, 183_184insG, 184_185insG, 185_186insG, 186_187insG, 272_273insG, 273_274insG, 274_275insG, 275_276insG, 276_277insG, 277_278insG, 278_279insG, 346G>A, 277delG, 362T>G, 369C>A, 3690>G, 375G>C, 382A>C, 386G>A, 3910>T, 427A>G, 485A>C, 484G>C, 484G>A, 488G>A, 500G>T, 502G>A, 509T>C, 515A>G, 5180>T, 5180>G, 520G>C, 5240>T, 527G>A, 5260>T, 5260>T, 528A>G, 533T>G, 542G>A, 455delC, 456delC, 457delC, 458delC, 460delC, 461delC, 462delC, 463delC, 464delC, 430delG, 431delG, 432delG, 433delG, 434delG, 435delG, 436delG, 429_430insGG, 430_431insGG, 431_432insGG, 432_433insGG, 433_434insGG, 434_435insGG, 435_436insGG, 436_437insGG, 437_438insGG, 438_439insGG, 553C>A, 554A>G, 559G>A, 5660>T, 582_584 del, 598G>T, 599G>A, 598G>T, 599G>C, 601A>C, 611T>G, 616G>A, 619G>C, 6220>T, 623T>A, 6460>T, 647G>A, 6460>T, 647G>C, 6580>T, 659G>A, 6640>T, 665G>A, 666delC, 667delC, 668delC, 669delC, 6790>T, 363_364insT, 429_430insG, 430_431insG, 431_432insG, 432_433insG, 433_434insG, 434_435insG, 435_436 insG, 436_437 insG, 437_438 insG, 459_460 insC, 460_461 insC, 461_462insC, 462_463 insC, 463_464 insC, 464_465 insC, 465_466 insC, 547_548 insC, 548_549insC, 549_550 insC, 551_552 insC, 552_553 insC, 553_554 insC, 650_651 insT, 716A>C, 2450>T, 733A>C, 746T>C, 7480>T, 7660>T, 769G>A, 782A>G, 782A>T, 781_793del, 548delC, 549delC, 550delC, 551delC, 552delC, 553delC, 8410>T, 853delG, 854delC, 855delC, 856delC, 8600>T, 862T>A, 872T>G, 878delG, 879delG, 880delG, 890T>C, 896del, 897delG, 898delC, 899delG, 900delG, 895_896 insGCC, 896_897 insGCC, 697_698insC, 698_699 insC, 699_700 insC, 700_701 insC, 832_833 insC, 833_834 insC, 834_835insC, 835_836 insC, 836_837 insC, 837_838 insC, 838_839 insC, 900_901 insA, 901_902insA, 902_903 insA, 903_904 insA, 903_904 insA, 904_905 insA, 905_906 insA, 906_907insA, 919delG, 944T>C, 965T>A, 996delG, 1007G>A, 10240>T, 1025A>G, 1060A>C, 1061delC, 1062delC, 1063delC, 1064delC, 1065delC, 1065delA, 1039G>A, 1117delG, 1118delG, 1119delG, 1120delG, 1121delG, 363_1128+201 del, 665insC, 666insC, 667insC, 668insC, 669insC, 918_919 insG, 928_929 insT, 1060_1061 insC, 1061_1062 insC, 1062_1063insC, 1063_1064 insC, 1064_1065 insC, and 1065_1066insC.
In one embodiment, a method of the invention comprises identifying one or more further HSV-2 TK mutation selected from 1_742 del, 8_9 insT, 1000>T, 153T>C, 157T>A, 25delC, 43delC, 175G>C, 176G>C, 181G>T, 186A>C, 196T>C, 180delG, 181delG, 182delG, 183delG, 214G>A, 2150>G, 219delG, 220delG, 221delG, 222delG, 2810>T, 280delG, 302T>G, 314A>C, 363_1128+204 del, 391A>C, 398A>T, 409G>T, 411C>A, 4110>G, 472T>C, 5290>T, 544A>G, 545G>A, 545G>C, 549G>A, 549G>C, 549G>U, 450delC, 451delC, 452delC, 463delC, 464delC, 465delC, 466delC, 467delC, 470delC, 471delC, 472delC, 519delC, 520delC, 521delC, 433delG, 434delG, 435delG, 436delG, 437delG, 438delG, 439delG, 416delT, 417delT, 418delT, 419delT, 482delT, 483delT, 484delT, 485delT, 432_433 insGG, 433_434 insGG, 434_435 insGG, 435_436 insGG, 436_437 insGG, 437_438insGG, 438_439 insGG, 439_440 insGG, 602G>A, 649G>A, 658A>T, 658A>C, 6640>T, 669G>A, 111_127 del, 365_366 insT, 551delC, 552delC, 553delC, 554delC, 555delC, 556delC, 550_551 insC, 551_552 insC, 552_553 insC, 553_554 insC, 554_555 insC, 555_556insC, 556_557insC, 585_586insC, 586_587 insC, 587_588 insC, 588_589 insC, 586_587 insC, 587_588 insC, 588_589 insC, 589_590insC, 590_591insC, 433delG, 434delG, 435delG, 436delG, 437delG, 438delG, 439delG432_433 insG, 433_434 insG, 434_435 insG, 435_436insG, 436_437insG, 437_438insG, 438_439insG, 439_440 insG, 439_440 insG, 440_441insG, 441_442 insG, 442_443 insG, 625_626 insT, 626_627 insT, 627_628 insT, 720T>G, 720T>A, 773_774 insG, 773_774 insG, 774_775 insG, 775_776 insG, 551_556 del, 551delC, 552delC, 553delC, 554delC, 555delC, 586delC, 587delC, 588delC, 589delC, 590delC, 651_654 del, 779delG, 780delG, 781delG, 782delG, 8140>G, 815G>T, 8140>T, 820G>A, 820G>C, 8210>G, 8630>T, 862_863 del, 921_922 insA, 999_1000 insC, 1000_1001insC, 1001_1002insC, 1002_1003insC, 1007G>A, 821_833del, 809delC, 810delC, 811delC, 812delC, 816delC, 817delC, 818delC, 819delC, 814_826 del, 856delG, 923delT, 924delT, 925delT, 926delT, and 927delT.
In one embodiment, a method of the invention comprises identifying one or more further HSV-1 DNA pol mutation selected from 1_742 del, 8_9 insT, 100C>T, 153T>C, 157T>A, 25delC, 43delC, 175G>C, 176G>C, 181G>T, 186A>C, 196T>C, 180delG, 181delG, 182delG, 183delG, 214G>A, 215C>G, 219delG, 220delG, 221delG, 222delG, 281C>T, 280delG, 302T>G, 314A>C, 363_1128+204 del, 391A>C, 398A>T, 409G>T, 411C>A, 411C>G, 472T>C, 529C>T, 544A>G, 545G>A, 545G>C, 549G>A, 549G>C, 549G>U, 450delC, 451delC, 452delC, 463delC, 464delC, 465delC, 466delC, 467delC, 470delC, 471delC, 472delC, 519delC, 520delC, 521delC, 433delG, 434delG, 435delG, 436delG, 437delG, 438delG, 439delG, 416delT, 417delT, 418delT, 419delT, 482delT, 483delT, 484delT, 485delT, 432_433 insGG, 433_434 insGG, 434_435 insGG, 435_436 insGG, 436_437insGG, 437_438insGG, 438_439insGG, 439_440insGG, 602G>A, 649G>A, 658A>T, 658A>C, 664C>T, 669G>A, 111_127 del, 365_366 insT, 551delC, 552delC, 553delC, 554delC, 555delC, 556delC, 550_551 insC, 551_552 insC, 552_553 insC, 553_554insC, 554_555insC, 555_556insC, 556_557insC, 585_586insC, 586_587 insC, 587_588 insC, 588_589 insC, 586_587 insC, 587_588 insC, 588_589 insC, 589_590insC, 590_591insC, 433delG, 434delG, 435delG, 436delG, 437delG, 438delG, 439delG432_433 insG, 433_434 insG, 434_435 insG, 435_436 insG, 436_437 insG, 437_438insG, 438_439insG, 439_440 insG, 439_440 insG, 440_441insG, 441_442insG, 442_443insG, 625_626insT, 626_627insT, 627_628insT, 720T>G, 720T>A, 773_774 insG, 773_774 insG, 774_775insG, 775_776insG, 551_556del, 551delC, 552delC, 553delC, 554delC, 555delC, 586delC, 587delC, 588delC, 589delC, 590delC, 651_654 del, 779delG, 780delG, 781delG, 782delG, 814C>G, 815G>T, 814C>T, 820G>A, 820G>C, 821C>G, 8630>T, 862_863 del, 921_922 insA, 999_1000 insC, 1000_1001 insC, 1001_1002 insC, 1002_1003insC, 1007G>A, 821_833 del, 809delC, 810delC, 811delC, 812delC, 816delC, 817delC, 818delC, 819delC, 814_826 del, 856delG, 923delT, 924delT, 925delT, 926delT, and 927delT.
In one embodiment, a method of the invention comprises identifying one or more further HSV-2 DNA pol mutation selected from 748G>C, 919G>A, 1882C>T, 2033A>G, 2049_2056insGAAGAC, 2170G>A, 2171C>T, 2173A>G, 2186G>A, 2194C>A, 2347C>A, 2353G>A, 2485C>A, 2548C>A, 2729T>C, 2734G>A, 2735A>T, and 2744C>T.
In a preferable embodiment, the presence of said one or more further HSV mutation (e.g. further HSV-1 TK mutation, further HSV-2 TK mutation, further HSV-1 DNA pol mutation and/or further HSV-2 DNA pol mutation) is indicative of the presence of an antiviral drug-resistant HSV. In one embodiment, the presence of said one or more HSV mutation confirms the presence of an antiviral drug-resistant HSV.
In a preferable embodiment, the absence of said one or more further HSV mutation (e.g. further HSV-1 TK mutation, further HSV-2 TK mutation, further HSV-1 DNA pol mutation and/or further HSV-2 DNA pol mutation) is indicative of the absence of an antiviral drug-resistant HSV. In one embodiment, the absence of said one or more HSV mutation confirms the absence of an antiviral drug-resistant HSV.
The presence or absence of said one or more further HSV mutation may be identified either within (i.e. constituting a step of) or externally to methods of the invention. In one embodiment, the methods of the invention comprise a step of identifying the presence or absence of said one or more further HSV mutation. The presence or absence of said one or more further HSV mutation may have been previously identified. In one embodiment, the presence or absence of said one or more further HSV mutation is identified externally to methods of the invention.
The person skilled in the art understands the nomenclature used to describe the mutations (e.g. nucleotide mutations) and polymorphisms (e.g. polypeptide polymorphisms) which are described herein. Nucleotide positions of TK and DNA pol genes are numbered relative to the first nucleotide of the coding sequence of the respective genes, wherein the first nucleotide of the coding sequence (e.g. the ‘adenine’, A, of the ATG start codon) corresponds to position 1. Preferably, the nucleotide positions of HSV-1 TK are numbered relative to the first nucleotide of SEQ ID NO: 1 (e.g. a reference sequence); the nucleotide positions of HSV-1 DNA pol are numbered relative to the first nucleotide of SEQ ID NO: 3 (e.g. a reference sequence); the nucleotide positions of HSV-2 TK are numbered relative to the first nucleotide of SEQ ID NO: 5 (e.g. a reference sequence); and/or the nucleotide positions of HSV-2 DNA pol are numbered relative to the first nucleotide of SEQ ID NO: 7 (e.g. a reference sequence).
Advantageously, diagnosis of infection and the severity of drug resistance can be made by the identification of a mutation in a TK or DNA pol sequence (e.g. via DNA sequencing or sequencing of the polypeptide sequence). The present method of diagnosis is objective and is highly accurate. It may be performed by non-experts with routine training in molecular techniques in a short period of time. The method of the invention may be used for diagnosing infection of a subject with an antiviral drug-resistant HSV. In a preferable embodiment, the presence or absence of one or more of said HSV mutations is identified in an isolated sample obtained from a subject.
Thus, in one aspect there is provided a method for diagnosing an infection with an antiviral drug-resistant herpes simplex virus (e.g. HSV-1 and/or HSV-2) in a subject, comprising:
In a preferable embodiment, said thymidine kinase (TK) mutation is selected from 250G>A (HSV-2), 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1). In a preferable embodiment, said DNA polymerase (DNA pol) mutation is selected from 1882C>G (HSV-2), 2405T>G (HSV-1), 2500G>T (HSV-1), 2515A>G (HSV-1), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and 2895_2896insT (HSV-1).
The term “diagnosis” as used herein encompasses identification, confirmation and/or characterisation of an antiviral drug-resistant HSV infection. Methods of diagnosis according to the invention are useful to confirm the existence of an infection. Methods of diagnosis are also useful in methods for assessment of clinical screening, prognosis, choice of therapy, evaluation of therapeutic benefit, i.e. for drug screening and drug development. Efficient diagnosis allows rapid identification of the most appropriate treatment (thus lessening unnecessary exposure to harmful drug side effects), and reducing relapse rates.
TK and/or DNA pol mutations described herein also find utility in the detection of antiviral drug-resistant HSV in e.g. non-patient samples, such as those obtained from medical equipment, surgical devices, or environmental samples.
In one embodiment, the method comprises detecting the presence or absence of one or more of said HSV mutations in a database of HSV mutations (e.g. TK and/or DNA pol mutations) associated with antiviral drug resistance. Suitably, said database comprises the mutations described herein.
In a preferable embodiment, said database comprises phenotypic data associated with the HSV mutation, preferably wherein said data is data on the susceptibility and/or resistance of an HSV comprising a mutation described herein to an antiviral drug.
In another aspect, there is provided a method for determining prognosis of an infection with an antiviral drug-resistant HSV in a subject, comprising detecting the presence or absence of one or more HSV mutation described herein in a sample obtained from a subject. In such aspects, the presence of said one or more HSV mutation correlates with a poor prognosis, and the absence of said one or more HSV mutation correlates with a good prognosis.
The term “Herpes Simplex Virus” (HSV) as used herein suitably encompasses both HSV-1 and HSV-2. The TK and DNA pol genes are highly conserved across HSV-1 and HSV-2, such that a resistance-associated mutation in one virus may also cause resistance in the other virus when present in the corresponding position (e.g. the corresponding position of the polypeptide and/or nucleic acid sequence). Thus, in one embodiment the HSV is HSV-1. In another embodiment, the HSV is HSV-2.
The term “antiviral drug-resistant” means that the HSV demonstrates resistance to an antiviral drug which is typically used to treat an HSV infection. The term “resistant” encompasses both weak (e.g. intermediate) resistance and strong resistance to one or more antiviral drug. The presence of weak (intermediate) and/or strong resistance may be determined using a plaque reduction assay, as described below. The term “susceptible” (e.g. in the context of an “antiviral drug-susceptible HSV” means that the HSV is not resistant to one or more antiviral drug which is typically used to treat an HSV infection.
In a preferable embodiment, the antiviral drug is one or more selected from foscarnet, cidofovir, penciclovir and aciclovir, or a combination thereof.
In one embodiment, the antiviral drug is one or more selected from foscarnet, cidofovir, or a combination thereof.
In one embodiment, the antiviral drug is one or more selected from penciclovir and aciclovir, or a combination thereof.
In one embodiment, an antiviral drug resistant HSV has weak/intermediate resistance to an antiviral drug. In one embodiment, an antiviral drug resistant HSV has strong resistance to an antiviral drug.
An assessment of said “antiviral drug-resistance”, is demonstrated by reference to the accompanying Examples, and may be assessed using the methodology described in the Examples (e.g. Example 2). For example, Example 3 describes a plaque reduction assay, which measures the “1050” of a drug against an HSV.
An assessment of “antiviral drug-resistance” can be made using a “plaque reduction assay” as is understood by the person skilled in the art. The plaque reduction assay may be carried out e.g. as described in Mbisa, Jean Lutamyo (September 2013), Antiviral Resistance Testing. In: eLS. John Wiley & Sons, Ltd: Chichester, which is incorporated herein by reference.
By way of example, a “plaque reduction assay” may comprise:
The “IC50” value is the concentration of antiviral drug sufficient to reduce the percentage of cells infected with a HSV in the test sample relative to the number of cells infected with a HSV in the control sample by 50%. Likewise, the “1020”, “1040”, “1060”, “1080” and “1090” values mean the concentration of antiviral drug sufficient to reduce the percentage of cells infected with a HSV in the test sample relative to the number of cells infected with a HSV in the control sample by 20%, 40%, 60%, 80% or 90%, respectively.
The test sample and the control sample are typically incubated at step b. for at least one hour (e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 hours).
The “lower defined concentration” of step e. is typically at least 2 times lower (e.g. at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 times lower). Suitably each time steps a.-d. are repeated (i.e. in step e.), the defined concentration of the drug is lowered accordingly.
Certain IC50 values of antiviral drugs correlate with certain levels of resistance, as outlined below.
Thus, in one embodiment a method of the invention comprises determining the level of resistance of a detected antiviral drug resistant HSV to an antiviral drug (e.g. strong resistance, weak/intermediate resistance, or no resistance/susceptibility). This can inform an appropriate choice of drug treatment and/or dosage of drug required for treatment. For example, where a mutation correlating with weak resistance is detected, a higher dose and/or longer course of treatment may be chosen.
The term “resistance phenotype” means the level of resistance of an HSV comprising a mutation described herein to an antiviral drug. Suitably, a resistance phenotype is selected from weak (e.g. intermediate) and strong resistance. Preferably, said resistance phenotype is determined via a plaque reduction assay.
In HSV infected cells, nucleoside analogue drugs (e.g. acyclovir and penciclovir) are phosphorylated by viral thymidine kinase and subsequently converted by cellular kinases into the active metabolite (e.g. acyclovir triphosphate and penciclovir triphosphate), which competitively inhibits viral HSV polymerase by competing with natural nucleoside (e.g. deoxyguanosine triphosphate) substrate binding. As a result, herpes viral DNA synthesis and replication are selectively inhibited. Without wishing to be bound by theory, it is believed that a TK enzyme comprising a mutation/polymorphism as described herein has reduced kinase activity, such that a nucleoside analogue drug (e.g. acyclovir and penciclovir) is not effectively phosphorylated (or is not phosphorylated at all) to produce an active drug. Thus, in one embodiment a TK comprising a TK mutation/polymorphism as described herein has reduced kinase (e.g. phosphorylation) activity. Suitably, a TK comprising a TK mutation/polymorphism as described herein does not phosphorylate a nucleoside analogue drug (e.g. acyclovir and penciclovir). Antiviral drugs which do not require phosphorylation by TK (e.g. cidofovir and foscarnet) are believed to directly inhibit viral DNA polymerase activity. Suitably, a DNA pol comprising a DNA pol mutation/polymorphism as described herein is not inhibited by an antiviral drug (e.g. cidofovir and foscarnet). Docosanol (PubChem CID: 12620) is a further antiviral drug used to treat HSV. The helicase-primase (e.g. of HSV-1) inhibitor BAY 54-6322 (Chemical Formula: C20H21N3O3S2; IUPAC Name: N-methyl-N-[4-methyl-5-(methylsulfamoyl)-1,3-thiazol-2-yl]-2-(4-phenylphenyl)acetamide) is a further antiviral drug used to treat HSV. The helicase-primase (e.g. of HSV-1) inhibitor ASP2151, also known as Amenamevir (PubChem CID: 11397521; Molecular Formula: C24H26N4O5S) is a further antiviral drug used to treat HSV. The helicase-primase (e.g. of HSV-1) inhibitor BAY 57-1293, also known as pritelivir (PubChem CID: 491941; Molecular Formula: C18H18N4O3S2) is a further antiviral drug used to treat HSV.
In one embodiment, the antiviral drug is acyclovir (PubChem CID: 2022; CAS Registry Number: 59277-89-3). In one embodiment, an antiviral-drug resistant HSV (e.g. HSV-1 or HSV-2) has strong resistance to acyclovir when the IC50 of the drug is greater than 40 μM (e.g. greater than 50 μM, 65 μM, 80 μM, 95 μM, 110 μM, 125 μM or 140 μM). In one embodiment, an antiviral-drug resistant HSV-1 has weak (intermediate) resistance to acyclovir when the IC50 of the drug is between about 3-40 μM (e.g. between about 6.5-40 μM, 5-35 μM, 10-30 μM, or 15-25 μM). In one embodiment, an antiviral-drug resistant HSV-2 has weak (intermediate) resistance to acyclovir when the IC50 of the drug is between about 6.5-40 μM (e.g. between about 10-30 μM, or 15-25 μM). In one embodiment, a HSV-1 is susceptible (e.g. non-resistant) to acyclovir when the IC50 is less than 3 μM (e.g. less than 2.5 μM, 2 μM, 1.5 μM, 1 μM or 0.5 μM). In one embodiment, a HSV-2 is susceptible (e.g. non-resistant) to acyclovir when the IC50 is less than 6.5 μM (e.g. less than 5 μM, 4 μM, 3 μM, 2 μM or 1 μM). In a preferable embodiment, the IC50 is determined in (e.g. via) a plaque reduction assay.
In one embodiment, the antiviral drug is penciclovir (PubChem CID: 4725; CAS Registry Number: 39809-25-1). In one embodiment, the antiviral-drug resistant HSV (e.g. HSV-1 or HSV-2) has strong resistance to pencyclovir when the IC50 of the drug is greater than 40 μM (e.g. greater than 50 μM, 65 μM, 80 μM, 95 μM, 110 μM, 125 μM or 140 μM). In one embodiment, an antiviral-drug resistant HSV-1 has weak (e.g. intermediate) resistance to pencyclovir when the IC50 of the drug (e.g. as determined in a plaque reduction assay) is between about 10-40 μM (e.g. between about 15-35 μM, or 20-30 μM). In one embodiment, an antiviral-drug resistant HSV-2 has weak (e.g. intermediate) resistance to pencyclovir when the IC50 of the drug is between about 38-40 μM (e.g. about 39 μM). In one embodiment, a HSV-1 is susceptible (e.g. non-resistant) to pencyclovir when the IC50 is less than 10 μM (e.g. less than 8 μM, 6 μM, 4 μM, 2 μM or 0.5 μM). In one embodiment, a HSV-2 is susceptible (e.g. non-resistant) to pencyclovir when the IC50 is less than 38 μM (e.g. less than 30 μM, 25 μM, 20 μM, 15 μM or 10 μM). In a preferable embodiment, the IC50 is determined in (e.g. via) a plaque reduction assay.
In one embodiment, the antiviral drug is foscarnet (PubChem CID: 3415). In one embodiment, an antiviral-drug resistant HSV (e.g. HSV-1 or HSV-2) has strong resistance to foscarnet when the IC50 of the drug is greater than 400 μM (e.g. greater than 450 μM, 500 μM, 550 μM, 600 μM, 650 μM, 700 μM or 750 μM). In one embodiment, the antiviral-drug resistant HSV (e.g. HSV-1 or HSV-2) has weak resistance to foscarnet when the IC50 of the drug is between about 250-400 μM (e.g. between about 275-385 μM, 300-360 μM, or 325-335 μM). In one embodiment, a HSV is susceptible (e.g. non-resistant) to foscarnet when the IC50 is less than 250 μM (e.g. less than 200 μM, 150 μM, 100 μM, 50 μM or 10 μM). In a preferable embodiment, the IC50 is determined in (e.g. via) a plaque reduction assay.
In one embodiment, the antiviral drug is cidofovir (PubChem CID: 60613; CAS Registry Number: 113852-37-2). In one embodiment, an antiviral-drug resistant HSV (e.g. HSV-1 or HSV-2) has strong resistance to cidofovir when the IC50 of the drug is greater than 30 μM (e.g. greater than 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM or 100 μM). In one embodiment, an antiviral-drug resistant HSV (e.g. HSV-1 or HSV-2) has weak resistance to cidofovir when the IC50 of the drug is between about 24-30 μM (e.g. between about 25-29 μM, or 26-28 μM). In one embodiment, a HSV is susceptible (e.g. non-resistant) to cidofovir when the IC50 is less than 24 μM (e.g. less than 20 μM, 15 μM, 10 μM, 5 μM or 1 μM). In a preferable embodiment, the IC50 is determined in (e.g. via) a plaque reduction assay.
In a preferable embodiment, identifying the presence of a TK mutation described herein confirms the presence of a acyclovir and/or penciclovir drug-resistant HSV. Suitably, identifying the absence of a TK mutation described herein is indicative of the absence of a acyclovir and/or penciclovir drug-resistant HSV. In another preferable embodiment, identifying the presence of a DNA pol mutation described herein confirms the presence of a acyclovir, penciclovir, foscarnet and/or cidofovir resistant HSV. Suitably, identifying the absence of a DNA pol mutation described herein is indicative of the absence of acyclovir, penciclovir, foscarnet and/or cidofovir resistant HSV.
The terms “subject”, “individual” and “patient” are used interchangeably herein to refer to a mammalian subject. In one embodiment the “subject” is a human, a companion animal (e.g. a pet such as dogs, cats, and rabbits), livestock (e.g. pigs, sheep, cattle, and goats), and horses. In a preferable embodiment, the subject is a human. Suitably, the subject (e.g. human) is immunocompromised. An immunocompromised subject may be a pregnant subject, a subject comprising an alternative infection (e.g. a HIV or meningococcal infection), or a haemato-oncology patient (e.g. undergoing bone-marrow transplant therapy). Suitably, the subject is a human undergoing bone-marrow transplant therapy. In methods of the invention, the subject may not have been previously diagnosed as having an HSV infection. Alternatively, the subject may have been previously diagnosed as having an HSV infection. The subject may also be one who exhibits disease risk factors, or one who is asymptomatic for an HSV infection. The subject may also be one who is suffering from or is at risk of developing an HSV infection. Thus, in one embodiment, a method of the invention may be used to confirm the presence of an HSV infection (e.g. an infection with an antiviral drug-resistant HSV) in a subject. For example, the subject may previously have been diagnosed with ASD through analysis of symptoms that the subject presents. In one embodiment, the subject has been previously administered an antiviral drug.
An “identifying” step (e.g. of one or more HSV mutation) may be performed manually, for example, by analysing a nucleic acid and/or polypeptide sequence. An identifying step may also be performed using any suitable method known in the art. A mutation may be identified within either the nucleic acid sequence or the polypeptide sequence. In one embodiment, DNA sequencing (e.g. of a PCR fragment/amplicon) may be used. In one embodiment, a TK sequence or fragment thereof is amplified. In one embodiment, a DNA pol sequence or fragment thereof is amplified. Said fragment may be of any length, and suitably encompasses the regions of the gene in which one or more of said mutations are present (and optionally includes 3′ and/or 5′ untranslated regions). Thus, in one embodiment a TK fragment (e.g. amplicon) comprises at least about 0.1 kilobases (kb), 0.2 kb, 0.3 kb, 0.4 kb, 0.5 kb, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, 1 kb, 1.1 kb, or 1.2 kb. In one embodiment, a DNA pol fragment (e.g. amplicon) comprises at least about 0.1 kilobases (kb), 0.2 kb, 0.3 kb, 0.4 kb, 0.5 kb, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, 1 kb, 1.1 kb, 1.2 kb, or 1.3 kb.
In another embodiment, the identifying step utilises a nucleic acid probe specific for one of more TK and/or DNA pol mutation (e.g. for use in PCR detection). In one embodiment, the identifying step is performed by mass spectrometry (e.g. to detect a polymorphism in a polypeptide sequence resulting from a mutation described herein). In another embodiment, the identifying step is performed by way of an immunoassay. Said immunoassay may employ the use of one or more antibodies that bind to one or more mutein(s) comprising a polymorphism described herein. Thus, in one aspect, there is provided an antibody composition for use in detecting one or more HSV mutation/polymorphism described herein. In another aspect, there is provided an antibody composition for use in the diagnosis of an infection with an antiviral-drug resistant HSV in a subject, wherein said antibody composition binds one or more mutein(s) comprising a polymorphism described herein. In another embodiment, the detecting step is performed by Western Blot or Enzyme activity assay. A method of the invention may comprise the use of a kit of the invention or a part thereof. For example, the method may comprise the use of a reagent described herein (preferably a nucleic acid PCR primer).
The methods or uses of the invention may encompass the detection of a HSV comprising a mutation described herein. The methods or uses of the invention may also encompass detecting no HSV comprising a mutation as described herein.
A method of the invention preferably involves the use of an algorithm or other data-driven combinatorial approach. In one embodiment, the identified mutations (e.g. TK and/or DNA pol mutations) are entered into an algorithm (e.g. as part of a computer software programme which may be provided in one aspect of the present invention), and said algorithm indicates whether an antiviral drug-resistant HSV is present, preferably by indicating whether the mutation is associated with antiviral drug resistance. In a preferable embodiment, said algorithm additionally indicates whether said mutation confers weak/intermediate or strong resistance to an antiviral drug.
In one embodiment, the algorithm interacts with a database comprising one or more HSV mutation. In a preferable embodiment, said database further comprises phenotypic data associated with one or more HSV mutation, preferably wherein said data is data on antiviral drug susceptibility/resistance. In one embodiment, the algorithm provides as an output the level (e.g. magnitude) of resistance associated with one or more HSV mutation, wherein said level of resistance is selected from one or more of weak/intermediate resistance, strong resistance and susceptibility (e.g. not resistant to one or more antiviral drug).
In one aspect, there is provided software adapted to provide an algorithm or diagnostic method of the invention. The invention also extends to a processor adapted to provide said software, algorithm and/or diagnostic method.
The skilled person will appreciate that any suitable algorithm can be used (including any one of the algorithms described herein). In one embodiment, the algorithm is an interpretation algorithm. In one embodiment, the algorithm is a machine learning algorithm. In one embodiment the algorithm is one or more selected from: Random Forests, logistic regression, ensemble classifier, Support Vector Machines (SVMs), general linear models (GLM), and GLMNET.
In one embodiment, the identifying (e.g. identification) steps of methods of the present invention comprise using a diagnostic algorithm configured to diagnose the presence or absence of an infection with an antiviral drug-resistant HSV (or to detect the presence or absence of an antiviral drug-resistant HSV), optionally wherein the diagnostic algorithm is trained on the mutations present in an antiviral drug-resistant HSV. In some embodiments, the algorithm predicts/provides the level of resistance (e.g. weak or strong resistance to an antiviral drug) associated with a mutation. Preferably, the algorithm is trained on the corresponding resistance phenotype for one or more of said mutation. Said embodiment can be applied to other methods such as the methods for determining prognosis.
Similar methods for identifying a therapy suitable for treating an antiviral drug-resistant HSV, and monitoring the efficacy of an antiviral drug-resistant HSV infection therapy are also provided.
In embodiments related to methods for identifying a therapy for an antiviral drug-resistant HSV, a candidate therapeutic is identified as suitable for treating an antiviral drug-resistant HSV infection when the viral load of said HSV comprising said one or more mutation in the test sample is lower than the viral load of a HSV comprising said one or more mutation in a control sample, wherein the control is incubated in the absence of an antiviral drug; and/or said candidate therapy is identified as not suitable for treating an antiviral drug-resistant HSV infection when the viral load of said HSV comprising said one or more mutation in the test sample is the same as (or greater than) the viral load of a HSV comprising said one or more mutation in a control sample, wherein the control is incubated in the absence of an antiviral drug.
A “sample” for use in the present invention is any sample that could comprises a HSV (e.g. antiviral drug-resistant HSV) or fragment thereof. Suitably, said sample may be isolated from a subject suspected of having an infection with an antiviral-drug resistant HSV. In some embodiments, the sample is isolated from a subject diagnosed as having an HSV infection. Suitably, a sample may be selected from one or more of a lesion, bodily fluid isolated from a lesion, blood, urine, eye fluid, lymphatic fluid, saliva, synovial fluid, seminal fluid, cerebrospinal fluid, sebaceous secretions, and/or sputum. In one embodiment, the sample is a viral suspension, optionally suspended in a suitable viral transport medium (e.g. Universal Transport Medium from Copan). In one embodiment, the sample is obtained from surgical or other medical equipment. In one embodiment, the sample is an environmental sample (e.g. water, soil and/or sediment).
Preferably the sample is a lesion (e.g. herpetic lesion) or fragment thereof. The term “lesion” encompasses any abnormality of tissue and/or an organ present in a subject having or suspected of having a HSV infection. Suitably, the sample is bodily fluid isolated from a lesion.
A key advantage to using a lesion or fragment thereof in methods of the present invention is that these sample are readily obtainable from a subject suspected of having an infection with an antiviral-drug resistant HSV and can be obtained using minimally invasive techniques (e.g. by contacting the lesion with a swab or other collecting device).
In one embodiment, a sample may be processed to isolate an HSV from a sample prior to detecting the presence or absence of an HSV comprising a mutation as described herein. In a preferable embodiment, the HSV is cultured from a sample prior to a detecting step of methods of the invention. In another embodiment, the viral genome or fragment thereof is first amplified and subsequently inserted (e.g. ligated) into a suitable vector (e.g. via restriction enzymes or by homologous recombination). The recombinant vectors may be introduced into cells by transfection to produce recombinant HSV.
In one embodiment, the sample comprises a recombinant (e.g. in vitro recombinant) HSV. Suitably, a recombinant HSV comprises one or more mutations/polymorphisms (e.g. TK and/or DNA pol mutations/polymorphisms) described herein.
In one aspect there is provided a recombinant (e.g. in vitro recombinant) HSV (e.g. HSV-1 and/or HSV-2) or fragment thereof comprising a mutation selected from: (i) a TK mutation selected from 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 376C>T (HSV-2), 146T>G (HSV-1), 250G>A (HSV-2), 253A>C (HSV-1), 256A>C (HSV-2), 363G>A (HSV-1), 366G>A (HSV-2), 497T>A (HSV-1), 500T>A (HSV-2), 558G>T (HSV-2), 715T>C (HSV-1), 718T>C (HSV-2), 935T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 276delG (HSV-2), 278delG (HSV-2), 279delG (HSV-2), 280delG (HSV-2), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1); and (ii) a DNA pol mutation selected from 1879C>G (HSV-1), 1882C>G (HSV-2), 2405T>G (HSV-1), 2420T>G (HSV-2), 2500G>T (HSV-1), 2515G>T (HSV-2), 2515A>G (HSV-1), 2530A>G (HSV-2), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), 2895_2896 insT, 2907_2908 insT (HSV-2), 2908_2909 insT (HSV-2), 2909_2910 insT (HSV-2) and 2910_2911 insT (HSV-2).
In a preferable embodiment, said thymidine kinase (TK) mutation is selected from 250G>A (HSV-2), 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1). In a preferable embodiment, said DNA polymerase (DNA pol) mutation is selected from 1882C>G (HSV-2), 2405T>G (HSV-1), 2500G>T (HSV-1), 2515A>G (HSV-1), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and 2895_2896 insT (HSV-1).
As used herein, the term “mutation” refers to any change in a nucleic acid, preferably relative to a reference sequence. Suitably, a mutation reduces (e.g. prevents) the interaction between TK and/or DNA pol polypeptide encoded thereby with an antiviral drug. The term “polymorphism” refers to any change in the polypeptide sequence, preferably relative to a reference sequence. Suitably, a polymorphism reduces (e.g. prevents) the interaction between TK and/or DNA pol with an antiviral drug.
In one embodiment, the mutation is a single nucleotide polymorphism (SNP).
In one embodiment, a TK SNP is selected from 100C>T (HSV-1), 268C>T (HSV-2), 3730>T (HSV-1), 376C>T (HSV-2), 146T>G (HSV-1), 250G>A (HSV-2), 253A>C (HSV-1), 256A>C (HSV-2), 363G>A (HSV-1), 366G>A (HSV-2), 497T>A (HSV-1), 500T>A (HSV-2), 558G>T (HSV-2), 715T>C (HSV-1), 718T>C (HSV-2), 935T>C (HSV-1), and 938T>C (HSV-2). In one embodiment, a DNA pol SNP is selected from 18790>G (HSV-1), 18820>G (HSV-2), 2405T>G (HSV-1), 2420T>G (HSV-2), 2500G>T (HSV-1), 2515G>T (HSV-2), 2515A>G (HSV-1), and 2530A>G (HSV-2). In one embodiment, a TK SNP is selected from 1000>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 376C>T (HSV-2), 146T>G (HSV-1), 250G>A (HSV-2), 253A>C (HSV-1), 256A>C (HSV-2), 363G>A (HSV-1), 366G>A (HSV-2), 497T>A (HSV-1), 500T>A (HSV-2), 558G>T (HSV-2), 715T>C (HSV-1), 718T>C (HSV-2), 935T>C (HSV-1), and 938T>C (HSV-2); and a DNA pol SNP is selected from 18790>G (HSV-1), 18820>G (HSV-2), 2405T>G (HSV-1), 2420T>G (HSV-2), 2500G>T (HSV-1), 2515G>T (HSV-2), 2515A>G (HSV-1), and 2530A>G (HSV-2). In a preferably embodiment, a TK SNP is selected from 250G>A (HSV-2), 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), and 938T>C (HSV-2). The skilled person understands that the sign “>” means that the first nucleotide referred to is substituted with/for the second nucleotide referred to at the position referred to. For example, where the substitution mutation is 100C>T (HSV-1), this means that the cytosine (“C”) at position 100 (nucleotide number 100) of the TK nucleic acid sequence is substituted for thymine (“T”).
In one embodiment, the mutation is an insertion (e.g. of one or more nucleotides).
In one embodiment, a TK insertion is 437_438 insA (HSV-1). In one embodiment, a DNA pol insertion is one or more selected from 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), 2895_2896 insT (HSV-1), 2907_2908 insT (HSV-2), 2908_2909 insT (HSV-2), 2909_2910 T (HSV-2), and 2910_2911 T (HSV-2) (preferably 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and/or 2895_2896 insT (HSV-1)). In one embodiment, a TK insertion is 437_438 insA (HSV-1); and a DNA pol insertion is one or more selected from 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), 2895_2895 insT (HSV-1), 2907_2908 insT (HSV-2), 2908_2909 insT (HSV-2), 2909_2910 insT (HSV-2) and 2910_2911 insT (HSV-2) (preferably 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and/or 2895_2896 insT (HSV-1)). The skilled person understands that the sign “ins” as used herein in the context of an insertion mutation means that a nucleotide(s) is inserted within the TK/DNA pol nucleic acid sequence between the positions referred to. For example, where the TK insertion mutation is 437_438 insA, this means that the nucleotide adenine (“A”) has been inserted after position 437 of the TK nucleic acid sequence (with said “A” now occupying position 438 of the mutated nucleic acid sequence).
In one embodiment, the mutation is a deletion (e.g. of one or more nucleotides).
In one embodiment, a TK deletion is selected from 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 276delG (HSV-2), 278delG (HSV-2), 279delG (HSV-2), 290delG (HSV-2), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1) (preferably 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and/or 885delC (HSV-1)). The skilled person understands the sign “del” as used herein in the context of a deletion polymorphism means that a nucleotide is deleted from the TK/DNA pol nucleic acid sequence at the position(s) referred to. For example, where the deletion mutation is 169delC, this means the cytosine (“C”) base has been deleted from position 169 of the TK nucleic acid sequence.
In one embodiment, the mutation causes a stop codon (e.g. early stop codon).
In one embodiment, a TK mutation causing a stop codon is selected from 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 376C>T (HSV-2), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 437_438 insA (HSV-1), 276delG (HSV-2), 278delG (HSV-2), 279delG (HSV-2), 280delG (HSV-2), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), and 461delC (HSV-2) (preferably 100C>T (HSV-1), 268C>T (HSV-2), 3730>T (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 437_438 insA (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), and/or 461delC (HSV-2)). In one embodiment, a DNA pol mutation causing a stop codon (e.g. early stop codon) is selected from 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1) and 2895_2896 insT. In one embodiment, a TK mutation causing a stop codon is selected from 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 3760>T (HSV-2), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 437_438 insA, 276delG (HSV-2), 278delG (HSV-2), 279delG (HSV-2), 280delG (HSV-2), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), and 461delC (HSV-2) (preferably 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 437_438 insA (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), and/or 461delC (HSV-2)); and a DNA pol mutation causing a stop codon is selected from 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895insT (HSV-1), and 2895_2896insT (HSV-1).
In one embodiment, the mutation causes a substitution polymorphism (e.g. an amino acid substitution) in the TK or DNA pol polypeptide sequence.
In one embodiment, a TK mutation causing a substitution polymorphism is selected from 146T>G (HSV-1), 363G>A (HSV-1), 366G>A (HSV-2), 497T>A (HSV-1), 500T>A (HSV-2), 715T>C (HSV-1), 718T>C (HSV-2), 250G>A (HSV-2), 253A>C (HSV-1), 256A>C (HSV-2), 558G>T (HSV-2), 935T>C (HSV-1), and 938T>C (HSV-2) (preferably 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 715T>C (HSV-1), 250G>A (HSV-2), 558G>T (HSV-2), and/or 938T>C (HSV-2). In one embodiment, a DNA pol mutation causing a substitution polymorphism is selected from 18790>G (HSV-1), 18820>G (HSV-2), 2405T>G (HSV-1), 2420T>G (HSV-2), 2500G>T (HSV-1), 2515G>T (HSV-2), 2515A>G (HSV-1), and 2530A>G (HSV-2) (preferably 18820>G (HSV-2), 2405T>G (HSV-1), 2500G>T (HSV-1), and/or 2515A>G (HSV-1)). In one embodiment, a TK mutation causing a substitution polymorphism is selected from 146T>G (HSV-1), 363G>A (HSV-1), 366G>A (HSV-2), 497T>A (HSV-1), 500T>A (HSV-2), 715T>C (HSV-1), 718T>C (HSV-2), 250G>A (HSV-2), 253A>C (HSV-1), 256A>C (HSV-2), 558G>T (HSV-2), 935T>C (HSV-1), and 938T>C (HSV-2) (preferably 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 715T>C (HSV-1), 250G>A (HSV-2), 558G>T (HSV-2), and/or 938T>C (HSV-2); and a DNA pol mutation causing a substitution polymorphism is selected from 18790>G (HSV-1), 18820>G (HSV-2), 2405T>G (HSV-1), 2420T>G (HSV-2), 2500G>T (HSV-1), 2515G>T (HSV-2), 2515A>G (HSV-1), and 2530A>G (HSV-2) (preferably 18820>G (HSV-2), 2405T>G (HSV-1), 2500G>T (HSV-1), and/or 2515A>G (HSV-1)).
In one embodiment, the mutation causes a frameshift (e.g. in the remainder of the coding sequence). In one embodiment, a TK mutation causing a frameshift is selected from 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1)), In one embodiment, a TK mutation causing a frameshift is selected from 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1).
In one embodiment, an HSV-1 TK mutation is one or more selected from 100C>T, 373C>T, 146T>G, 253A>C, 363G>A, 497T>A, 715T>C, 935T>C, 437_438 insA, 169delC, 170delC, 171delC, 172delC, 881delC, 882delC, 883delC, 884delC, and 885delC (preferably 100C>T, 373C>T, 146T>G, 363G>A, 497T>A, 715T>C, 437_438 insA, 169delC, 170delC, 171delC, 172delC, 881delC, 882delC, 883delC, 884delC, and/or 885delC). In one embodiment, an HSV-1 TK mutation is one or more selected from 100C>T, 146T>G, 363G>A, 373C>T, 497T>A, 715T>C, 169delC, 170delC, 171delC, 172delC, 437_438 insA, 881delC, 882delC, 883delC, 884delC, and 885delC. In one embodiment, an HSV-1 TK mutation is one or more selected from 146T>G, 497T>A, 169delC, 170delC, 171delC, 172delC, 437_438 insA, 881delC, 882delC, 883delC, 884delC, and 885delC.
In one embodiment, an HSV-2 TK mutation is one or more selected from 268C>T, 376C>T, 250G>A, 256A>C, 366G>A, 500T>A, 558G>T, 718T>C, 938T>C, 276delG, 278delG, 279delG, 280delG, 458delC, 459delC, 460delC, and 461delC (preferably 268C>T, 250G>A, 558G>T, 938T>C, 458delC, 459delC, 460delC, and/or 461delC). In one embodiment, an HSV-2 TK mutation is one or more selected from 250G>A, 256A>C, 268C>T, 558G>T, 938T>C, 1094C>T, 276delG, 278delG, 279delG, 280delG, 458delC, 459delC, 460delC, and 461delC (preferably 250G>A, 268C>T, 558G>T, 938T>C, 458delC, 459delC, 460delC, and/or 461delC). In one embodiment, an HSV-2 TK mutation is one or more selected from 256A>C, 558G>T, 938T>C, 1094C>T, 276delG, 278delG, and 279delG (preferably 558G>T, and/or 938T>C).
In one embodiment, a TK mutation is one or more selected from 100C>T, 373C>T, 146T>G, 253A>C, 363G>A, 497T>A, 715T>C, 935T>C, 437_438 insA, 169delC, 170delC, 171delC, 172delC, 881delC, 882delC, 883delC, 884delC, and 885delC (preferably 100C>T, 373C>T, 146T>G, 363G>A, 497T>A, 715T>C, 437_438 insA, 169delC, 170delC, 171delC, 172delC, 881delC, 882delC, 883delC, 884delC, and/or 885delC) wherein the HSV is HSV-1 and the TK mutation is one or more selected from 268C>T, 376C>T, 250G>A, 256A>C, 366G>A, 500T>A, 558G>T, 718T>C, 938T>C, 276delG, 278delG, 279delG, 280delG, 458delC, 459delC, 460delC, and 461delC (preferably 268C>T, 250G>A, 558G>T, 938T>C, 458delC, 459delC, 460delC, and/or 461delC) wherein the HSV is HSV-2.
In one embodiment, a HSV-1 DNA pol mutation is one or more selected from 1879C>G, 2405T>G, 2500G>T, 2515A>G, 2892_2893 insT, 2893_2894 insT, 2894_2895 insT, and 2895_2896 insT (preferably 2405T>G, 2500G>T, 2515A>G, 2892_2893 insT, 2893_2894 insT, 2894_2895 insT, and/or 2895_2896 insT). In one embodiment, an HSV-1 DNA pol mutation is one or more selected from 2405T>G, 2500G>T, 2515A>G, 2892_2893 insT, 2893_2894 insT, 2894_2895 insT, and 2895_2896 insT. In one embodiment, an HSV-1 DNA pol mutation is one or more selected from 2500G>T, 2515A>G, 2892_2893 insT, 2893_2894 insT, 2894_2895 insT, and 2895_2896 insT.
In one embodiment, a HSV-2 DNA pol mutation is one or more selected from 18820>G, 2420T>G, 2515G>T, 2530A>G, 2907_2908 insT, 2908_2909 insT, 2909_2910 insT, and 2910_2911insT (preferably 18820>G). In one embodiment, a HSV-2 DNA pol mutation is one or more selected from 2420T>G, 2515G>T, 2530A>G, 2907_2908 insT, 2908_2909 insT, 2909_2910insT, 2910_2911insT. In one embodiment, an HSV-2 DNA pol mutation is 18820>G.
In one embodiment, the DNA pol mutation is one or more selected from 18790>G, 2405T>G, 2500G>T, 2515A>G, 2892_2893 insT, 2893_2894 insT, 2894_2895 insT, and 2895_2896 insT preferably 2405T>G, 2500G>T, 2515A>G, 2892_2893 insT, 2893_2894 insT, 2894_2895 insT, and/or 2895_2896 insT) wherein the HSV is HSV-1 and the DNA pol mutation is one or more selected from 18820>G, 2420T>G, 2515G>T, 2530A>G, 2907_2908 insT, 2908_2909 insT, 2909_2910insT and 2910_2911insT (preferably 18820>G) wherein the HSV is HSV-2.
An HSV comprising said one or more mutation and/or may be referred to as a modified HSV, having a modified nucleic acid and/or amino acid sequence relative to a reference (wild-type) HSV sequence.
In a preferable embodiment, one or more (preferably all) of the mutations described herein are not present in a reference (e.g. wild-type) HSV sequence. Preferably, a HSV (e.g. HSV-1 or HSV-2) comprising the reference sequence is not resistant to an antiviral drug as described herein. The nucleic acid and polypeptide sequences of preferable reference HSV sequences are publically available. In one embodiment, the reference HSV-1 sequence (e.g. publically available sequence) is accessible on GenBank with accession number JN555585.1. The TK nucleic acid sequence, the TK polypeptide sequence, the DNA pol nucleic sequence and the DNA pol polypeptide sequence of this HSV-1 sequence corresponds to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, respectively. In one embodiment, the reference HSV-2 sequence (e.g. publically available sequence) is accessible on GenBank with accession number JN561323.2. The TK nucleic acid sequence, the TK polypeptide sequence, the DNA pol nucleic acid sequence and the DNA pol polypeptide sequence of this HSV-2 sequence corresponds to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, respectively.
In one embodiment, a reference HSV-1 TK nucleic acid comprises (or consists of) the sequence of SEQ ID NO: 1, or a sequence having at least 90% sequence identity thereto, suitably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In one embodiment, a reference HSV-1 TK polypeptide comprises (or consists of) the sequence of SEQ ID NO: 2, or a sequence having at least 90% sequence identity thereto, suitably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In one embodiment, a reference HSV-1 DNA pol nucleic acid comprises (or consists of) the sequence of SEQ ID NO: 3, or a sequence having at least 90% sequence identity thereto, suitably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In one embodiment, a reference HSV-1 DNA pol polypeptide comprises (or consists of) the sequence of SEQ ID NO: 4, or a sequence having at least 90% sequence identity thereto, suitably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In one embodiment, a reference HSV-2 TK nucleic acid comprises (or consists of) the sequence of SEQ ID NO: 5, or a sequence having at least 90% sequence identity thereto, suitably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In one embodiment, a reference HSV-2 TK polypeptide comprises (or consists of) the sequence of SEQ ID NO: 6, or a sequence having at least 90% sequence identity thereto, suitably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In one embodiment, a reference HSV-2 DNA pol nucleic acid comprises (or consists of) the sequence of SEQ ID NO: 7, or a sequence having at least 90% sequence identity thereto, suitably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In one embodiment, a reference HSV-2 DNA pol polypeptide comprises (or consists of) the sequence of SEQ ID NO: 8, or a sequence having at least 90% sequence identity thereto, suitably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
A reference HSV-1 TK polypeptide may comprise the sequence obtainable with a UniProt accession number selected from G8HBD6, C0L307, C0L309, A0A0U2UZK2, 12FEY2, C0L308, K4JR12, and C0L314. A reference HSV-1 DNA pol polypeptide may comprise the sequence obtainable with a UniProt accession number selected from G8HBE4, E1B1U3, P04293, I1YAD1, I1YAC1, 11YA98, A0A1C3K996, and E1B1Y8. A reference HSV-2 TK polypeptide may comprise the sequence obtainable with a UniProt accession number selected from P89446, Q6L709, E1B1R4, E1B1Y2, A0A0B4WW69, A0A0K0KND3, E1B1Y1, and A0A0B4WUV0. A reference HSV-2 DNA pol polypeptide may comprise the sequence obtainable with a UniProt accession number selected from P89453, E1B1W8, E1B202, E1B1X6, E1B212, A0A1U9ZMP4, A0A1U9ZLW2, I1YAG2 and A0A0K0KNB4.
The reference HSV sequence may be obtained either within (i.e. constituting a step of) or externally to methods of the invention. In one embodiment, the methods of the invention comprise a step of obtaining a reference HSV sequence, preferably wherein the reference HSV sequence is the sequence of a HSV which is not resistant to an antiviral drug described herein. In one embodiment, the reference HSV sequence is/are obtained externally to the method of the invention and accessed during the detecting and/or identifying step of the present invention.
In one embodiment, the term “at least one” when used in the context of a TK mutation described herein means at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or all of the TK polymorphisms. In one embodiment, the term “at least one” when used in the context of a DNA pol mutation described herein means at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 10, or all of the DNA pol mutations.
A mutation causing antiviral drug resistance as described herein is distinct from “mutation not associated with antiviral drug resistance” (e.g. a “non-antiviral drug resistance associated mutation” or “non-antiviral drug resistance associated polymorphism”) which does not cause antiviral drug resistance. A HSV may comprise a multitude of “mutations not associated with antiviral drug resistance”, many of which occur naturally and do not alter the structure and/or function of HSV polypeptides. The present inventors have identified a number of ‘natural’ mutations i.e. non-antiviral drug resistance associated. Furthermore, the present inventors have elucidated the phenotype of an HSV comprising one or more of said “mutations not associated with antiviral drug resistance”, namely susceptibility to an antiviral drug. A “mutation not associated with antiviral drug resistance” as described herein is one which does not result in resistance to an antiviral drug. Advantageously, the detection of such ‘natural’ polymorphism increases the robustness of the interpretation of a resistance-associated mutation (e.g. the interpretation of a database of resistance associated mutations, and algorithm for their detection). Advantageously, by providing such ‘natural’ polymorphisms, the present invention avoids the problem of false-positive detection of antiviral drug resistance.
A mutation not associated with antiviral drug resistance in the TK sequence of HSV-1 (with the corresponding/resulting amino acid substitution shown in parentheses) may be one or more selected from 110C>T (A37V), 205C>A (L69M), 1072A>C or 1072A>T (1358L), 574G>A (A192T), 766C>T (R256W).
A mutation not associated with antiviral drug resistance in the TK sequence of HSV-2 (with the corresponding/resulting amino acid substitution shown in parentheses) may be one or more selected from 100C>T (R34C), 373G>A (A125T), 639A>C or 639A>T (E213D), or 1094T>C (L365P) (preferably 373G>A, 639A>C or 639A>T, or 1094T>C).
A mutation not associated with antiviral drug resistance in the DNA pol sequence of HSV-1 (with the corresponding/resulting amino acid substitution shown in parentheses) may be one or more selected from 64G>A (G22R), 160A>G (T54A), 248A>C (D83A), 361G>A (G121S), 415G>A or 415G>C (G139R), 716C>T (S239L), 1255C>A (L4191), 2039A>C (E680A), 2042G>A (R681Q), 2249A>C (K750T), 2548G>C (E850Q), 2732G>A (S911N), 2741C>T (S914L), 2915C>T (A972V), 2954G>A (G985E), 2974G>A (E992K), 2977C>T (R993C), 3137A>C (N1046T), 3343G>A (A1115T), 3359A>C (E1120A), 3505G>A (A1169T), and 3595C>A (P1199T).
A mutation not associated with antiviral drug resistance in the DNA pol sequence of HSV-2 (with the corresponding/resulting amino acid substitution shown in parentheses) may be one or more selected from 520G>T (D174Y), 1339A>C or 1339A>T (M447L), 1481T>C (M494T), 2141A>G (H714R), 2281G>A (E761K), 2323C>T (R775C), 2325C>G (R775W), and 2326G>C (E776Q).
Alternatively or additionally (preferably additionally), a method of the invention comprises:
Alternatively or additionally (preferably additionally), a method of the invention comprises:
In one aspect, the invention provides a method for identifying a therapeutic suitable for treating an antiviral drug-resistant HSV (e.g. HSV-1 and/or HSV-2) infection, comprising:
In a preferable embodiment, said thymidine kinase (TK) mutation is selected from 250G>A (HSV-2), 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1). In a preferable embodiment, said DNA polymerase (DNA pol) mutation is selected from 1882C>G (HSV-2), 2405T>G (HSV-1), 2500G>T (HSV-1), 2515A>G (HSV-1), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and 2895_2896insT (HSV-1).
The skilled person understands that where the methods of the invention comprise a comparison step between two samples (e.g. between a “test sample” and a “control sample”) that conditions (e.g. assay conditions during the method) should be kept consistent. For example, the viral load (e.g. starting viral load) of the HSV in both the test sample and control sample should be the same, as should culture conditions, etc.
The viral load of a HSV comprising said one or more mutation in a control sample may be determined either within (i.e. constituting a step of) or externally to methods of the invention. In one embodiment, the methods of the invention comprise a step of incubating a control sample in the absence of an antiviral drug. In one embodiment, the concentration of a HSV comprising said one or more mutation in a control sample is obtained externally to the method of the invention and accessed during the comparison step of the present invention.
Another aspect provides a method for monitoring the efficacy of an HSV therapy in a subject infected with an antiviral drug-resistant HSV (e.g. HSV-1 and/or HSV-2), said method comprising:
c. determining the relative change in viral load of said HSV comprising said one or more mutation by comparing the viral load of HSV comprising said one or more mutation detected in step (b) with the viral load of a HSV comprising said one or more mutation in an isolated sample obtained from the subject at an earlier timepoint; and confirming the presence of efficacy when the viral load is decreased; and confirming the absence of efficacy when the viral load is increased (or unchanged).
In a preferable embodiment, said thymidine kinase (TK) mutation is selected from 250G>A (HSV-2), 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1). In a preferable embodiment, said DNA polymerase (DNA pol) mutation is selected from 1882C>G (HSV-2), 2405T>G (HSV-1), 2500G>T (HSV-1), 2515A>G (HSV-1), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and 2895_2896 insT (HSV-1).
The term “earlier timepoint” may refer to a timepoint of at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 52 weeks, at least 2 years, at least 3 years, or at least 4 years earlier.
In one aspect, there is provided an antiviral drug for use in a method for treating an infection of an antiviral drug-resistant HSV (e.g. HSV-1 and/or HSV-2), wherein said method comprises:
In a preferable embodiment, said thymidine kinase (TK) mutation is selected from 250G>A (HSV-2), 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1).
Suitably, the antiviral drug is selected from foscarnet, cidofovir drug, docosanol, BAY 54-6322, ASP2151 and BAY 57-1293.
In one aspect, there is provided an antiviral drug for use in a method for treating an infection of an antiviral drug-resistant HSV (e.g. HSV-1 and/or HSV-2), wherein said method comprises:
In a preferable embodiment, said DNA polymerase (DNA pol) mutation is selected from 1882C>G (HSV-2), 2405T>G (HSV-1), 2500G>T (HSV-1), 2515A>G (HSV-1), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and 2895_2896 insT (HSV-1).
In one aspect, there is provided use of a TK mutation selected from 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 376C>T (HSV-2), 146T>G (HSV-1), 250G>A (HSV-2), 253A>C (HSV-1), 256A>C (HSV-2), 363G>A (HSV-1), 366G>A (HSV-2), 497T>A (HSV-1), 500T>A (HSV-2), 558G>T (HSV-2), 715T>C (HSV-1), 718T>C (HSV-2), 935T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 276delG (HSV-2), 278delG (HSV-2), 279delG (HSV-2), 280delG (HSV-2), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1), or a combination thereof for:
In a preferable embodiment, said thymidine kinase (TK) mutation is selected from 250G>A (HSV-2), 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1).
In another aspect, there is provided use of a DNA pol mutation selected from 1879C>G (HSV-1), 1882C>G (HSV-2), 2405T>G (HSV-1), 2420T>G (HSV-2), 2500G>T (HSV-1), 2515G>T (HSV-2), 2515A>G (HSV-1), 2530A>G (HSV-2), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), 2895_2896 insT (HSV-1), 2907_2908 insT (HSV-2), 2908_2909 insT (HSV-2), 2909_2910 insT (HSV-2), and 2910_2911 insT (HSV-2), or a combination thereof for:
In a preferable embodiment, said DNA polymerase (DNA pol) mutation is selected from 1882C>G (HSV-2), 2405T>G (HSV-1), 2500G>T (HSV-1), 2515A>G (HSV-1), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and 2895_2896 insT (HSV-1).
In one aspect, there is provided use of an HSV mutation selected from a TK polymorphism mutation from 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 376C>T (HSV-2), 146T>G (HSV-1), 250G>A (HSV-2), 253A>C (HSV-1), 256A>C (HSV-2), 363G>A (HSV-1), 366G>A (HSV-2), 497T>A (HSV-1), 500T>A (HSV-2), 558G>T (HSV-2), 715T>C (HSV-1), 718T>C (HSV-2), 935T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 276delG (HSV-2), 278delG (HSV-2), 279delG (HSV-2), 280delG (HSV-2), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1); and a DNA pol mutation selected from 1879C>G (HSV-1), 1882C>G (HSV-2), 2405T>G (HSV-1), 2420T>G (HSV-2), 2500G>T (HSV-1), 2515G>T (HSV-2), 2515A>G (HSV-1), 2530A>G (HSV-2), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), 2895_2896 insT (HSV-1), 2907_2908 insT (HSV-2), 2908_2909 insT (HSV-2), 2909_2910 insT (HSV-2), and 2910_2911 insT (HSV-2), or a combination thereof for:
In a preferable embodiment, said thymidine kinase (TK) mutation is selected from 250G>A (HSV-2), 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1). In a preferable embodiment, said DNA polymerase (DNA pol) mutation is selected from 1882C>G (HSV-2), 2405T>G (HSV-1), 2500G>T (HSV-1), 2515A>G (HSV-1), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and 2895_2896 insT (HSV-1).
In one embodiment, a method or use of the present invention comprises the step of recording on a suitable data carrier, the data obtained in the step of detecting the presence or absence said one or more HSV mutation.
In one aspect, there is provided a data carrier comprising the data obtained in the step of identifying one or more HSV mutation according to a method of the invention. In another aspect, there is provided data carrier comprising the data obtained in the step of detecting the presence or absence of said one or more HSV mutation according to a method of the invention for use in a method for diagnosing an infection with an antiviral drug-resistant HSV.
In another aspect, there is provided a kit comprising reagents for detecting the presence or absence of (e.g. identifying) one or more HSV mutation selected from: (i) a TK mutation selected from 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 376C>T (HSV-2), 146T>G (HSV-1), 250G>A (HSV-2), 253A>C (HSV-1), 256A>C (HSV-2), 363G>A (HSV-1), 366G>A (HSV-2), 497T>A (HSV-1), 500T>A (HSV-2), 558G>T (HSV-2), 715T>C (HSV-1), 718T>C (HSV-2), 935T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 276delG (HSV-2), 278delG (HSV-2), 279delG (HSV-2), 280delG (HSV-2), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1); and (ii) a DNA pol mutation selected from 1879C>G (HSV-1), 1882C>G (HSV-2), 2405T>G (HSV-1), 2420T>G (HSV-2), 2500G>T (HSV-1), 2515G>T (HSV-2), 2515A>G (HSV-1), 2530A>G (HSV-2), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), 2895_2896 insT (HSV-1), 2907_2908 insT (HSV-2), 2908_2909 insT (HSV-2), 2909_2910 insT (HSV-2), and 2910_2911 insT (HSV-2); and instructions for use of the same.
In a preferable embodiment, said thymidine kinase (TK) mutation is selected from 250G>A (HSV-2), 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1). In a preferable embodiment, said DNA polymerase (DNA pol) mutation is selected from 18820>G (HSV-2), 2405T>G (HSV-1), 2500G>T (HSV-1), 2515A>G (HSV-1), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and 2895_2896insT (HSV-1).
In one embodiment, the reagents of said kit are for detecting the presence or absence of one or more HSV mutation by DNA sequencing, sequence capture, mass spectrometry, Western Blot, Enzyme activity assay and/or Enzyme-Linked Immunosorbent Assay (ELISA) (e.g. preferably by DNA sequencing) (e.g. preferably by mass spectrometry).
The present inventors have also identified the polypeptide polymorphisms caused by the mutations described herein.
All aspects, embodiments and definitions relating to mutations (e.g. nucleic acid sequence mutations) are also applicable to the corresponding aspects/embodiments, wherein the “mutation” is simply replaced by the corresponding “polymorphism” (e.g. polypeptide polymorphism). Corresponding polymorphisms of preferable mutations are shown in Tables 1-3.
An HSV-2 DNA pol mutation of 18820>G results in a R628G polymorphism. Suitably, an HSV-2 comprising said mutation/polymorphism has weak/intermediate resistance to acyclovir.
In one aspect there is provided a method for detecting the presence or absence of an antiviral drug-resistant HSV (e.g. in a subject), comprising:
Optionally, a description of a polymorphism may be preceded by the sign p. to denote identification of a polymorphism in a polypeptide. For example, a polymorphism L49R may be referred to as p.L49R.
In one aspect, there is provided a method for treating an infection of an antiviral drug-resistant HSV in a subject, comprising:
In one embodiment, step b. comprises administering to said subject a drug selected from a foscarnet drug, cidofovir drug, a docosanol drug, BAY 54-6322, ASP2151 and BAY 57-1293.
In one aspect, there is provided a method for treating an infection of an antiviral drug-resistant HSV in a subject, comprising:
In one aspect, there is provided an antiviral drug for use in a method for treating an infection of an antiviral drug-resistant HSV (e.g. HSV-1 and/or HSV-2), wherein said method comprises:
In one embodiment, step b. comprises administering to said subject a drug selected from a foscarnet drug, cidofovir drug, a docosanol drug, BAY 54-6322, ASP2151 and BAY 57-1293.
In one aspect, there is provided an antiviral drug for use in a method for treating an infection of an antiviral drug-resistant HSV (e.g. HSV-1 and/or HSV-2), wherein said method comprises:
In another aspect, there is provided a kit comprising reagents for detecting the presence or absence of one or more HSV mutation selected from: (i) a TK polymorphism selected from Q34* (HSV-1), Q90* (HSV-2), Q125* (HSV-1), Q126* (HSV-2), L49R (HSV-1), E84K (HSV-2), M85L (HSV-1), M86L (HSV-2), M1211 (HSV-1), M1221 (HSV-2), I166N (HSV-1), I167N (HSV-2), Q186H (HSV-2), Y239H (HSV-1), Y240H (HSV-2), L312S (HSV-1), L313S (HSV-2), T183* (HSV-1), M85* (HSV-1), L98* (HSV-2), M183* (HSV-2), A294fs (HSV-1), P295fs (HSV-1), E296fs (HSV-1); and (ii) a DNA polymerase (DNA pol) polymorphism selected from R627G (HSV-1), R628G (HSV-2), L802R (HSV-1), L807R (HSV-2), A834S (HSV-1), A839S (HSV-2), T839A (HSV-1), T844A (HSV-2), 1966* (HSV-1), and 1971* (HSV-2); and instructions for use of the same.
In a preferable embodiment, said TK polymorphism selected from E84K (HSV-2), Q34* (HSV-1), Q90* (HSV-2), Q125* (HSV-1), L49R (HSV-1), M1211 (HSV-1), 1166N (HSV-1), Q186H (HSV-2), H214R (HSV-2), E146fs (HSV-1), D228* (HSV-1), Y239H (HSV-1), L313S (HSV-2), T183* (HSV-1), H58fs (HSV-1), M85* (HSV-1), P154fs (HSV-2), M183* (HSV-2), A294fs (HSV-1), P295fs (HSV-1), and E296fs (HSV-1). In a preferable embodiment, R628G (HSV-2), L802R (HSV-1), A834S (HSV-1), T839A (HSV-1), F965_I966insF (HSV-1), and 1966* (HSV-1).
The methods of the present invention encompass identifying a mutation in the nucleic acid of a HSV and/or a polymorphism in the polypeptide sequence of a HSV. As such, a mutation or polymorphism may be described either by the nucleic acid mutation or the resulting amino acid polymorphism. For example, a “611T>G” single nucleotide polymorphism in the nucleic acid sequence of a HSV-1 TK results in the amino acid polymorphism (e.g. substitution) “V204G” in the polypeptide sequence of said HSV-1 TK. Thus, in one embodiment a nucleic acid mutation is identified. In another embodiment, a polypeptide polymorphism is identified.
The polypeptide polymorphisms resulting from the mutations described herein are outlined below. Thus, the mutation may be detected either directly within a nucleotide sequence or within a polypeptide sequence (e.g. by inferring the presence or absence of a mutation by detecting the presence of absence of the resulting polypeptide polymorphism). All references to “identifying one or more HSV mutation” herein may be substituted for “identifying one or more HSV polymorphism”.
In one embodiment, the TK and/or DNA Pol polymorphism is a stop codon (e.g. early stop codon).
In one embodiment, a HSV-1 TK stop codon (e.g. early stop codon) polypeptide polymorphism is selected from Q34* (or Q34X), Q125* (or Q125X), M85* (or M85X), and T183* (or T183X). In one embodiment, a HSV-2 TK stop codon (e.g. early stop codon) polypeptide polymorphism is selected from Q126* (or Q126X), Q90* (or Q90X) and L98* (or L98X) (preferably Q126*).
In one embodiment, a HSV-1 DNA pol stop codon (e.g. early stop codon) polypeptide polymorphism is 1966* (or G966X).
In one embodiment, a HSV-1 TK stop codon (e.g. early stop codon) polypeptide polymorphism is selected from Q34* (or Q34X), Q125* (or Q125X), M85* (or M85X), and T183* (or T183X); a HSV-2 TK stop codon (e.g. early stop codon) polypeptide polymorphism is selected from Q126* (or Q126X), Q90* (or Q90X) and L98* (or L98X); and a HSV-1 DNA pol stop codon (e.g. early stop codon) polypeptide polymorphism is 1966* (or 1966X).
The skilled person understands that the asterix sign “*” (or, in alternative nomenclature, the “X” sign) denotes that the amino acid referred to is not expressed, as the codon encoding it is substituted with a stop codon (e.g. TAA, TAG, or TGA). For example, where the stop codon polymorphism is Q34* (or Q34X), this means that the glutamine (“Q”) at position 34 of the TK polypeptide sequence is not translated, and that translation of the polypeptide ceases after amino acid 33. As such, the TK polypeptide is truncated.
In one embodiment, the TK and/or DNA Pol polymorphism is a (amino acid) substitution.
In one embodiment, a HSV-1 TK substitution polymorphism is selected from L49R, M85L, M1211, 1166N, Y239H, and L312S (preferably L49R, M1211, 1166N, and/or Y239H). In one embodiment, a HSV-2 TK substitution polymorphism is selected from E84K, M86L, M1221, 1167N, Q186H, Y240H, and L313S (preferably E84K, Q186H, and/or L313S).
In one embodiment, a HSV-1 DNA pol substitution polymorphism is selected from R627G, L802R, A834S, and T839A (preferably L802R, A834S, and/or T839A). In one embodiment, a HSV-2 DNA pol substitution polymorphism is selected from R628G, L807R, A839S, and T844A (preferably R628G).
In one embodiment, a HSV-1 TK substitution polymorphism is selected from L49R, M85L, M1211, 1166N, Y239H, and L312S (preferably L49R, M1211, 1166N, and/or Y239H); a HSV-2 TK substitution polymorphism is selected from E84K, M86L, M1221, 1167N, Q186H, Y240H, and L313S preferably E84K, Q186H, and/or L313S); a HSV-1 DNA pol substitution polymorphism is selected from R627G, L802R, A834S, and T839A (preferably L802R, A834S, and/or T839A); and a HSV-2 DNA pol substitution polymorphism is selected from R628G, L807R, A839S, and T844A (preferably R628G).
The skilled person understands the nomenclature used herein in the context of a substitution polymorphism. For example, where the substitution polymorphism is L49R, this means the lysine (“L”) amino acid at position 49 of the TK polypeptide is substituted for arginine (“R”).
In one embodiment, the TK and/or DNA Pol polymorphism is a frameshift.
In one embodiment, a HSV-1 TK frameshift polymorphism is selected from E146FS, H58fs, and E296fs. In one embodiment, a HSV-1 TK frameshift polymorphism is selected from A294fs, P295fs, and E296fs.
The sign “fs” denotes a frameshift (e.g. throughout the remainder of the polypeptide sequence) beginning at the amino acid referred to.
In a preferable embodiment, one or more (preferably all) of the polymorphisms described herein are not present in a reference (e.g. wild-type) HSV sequence.
Amino acids relevant to the present invention are outlined below. The one letter code for said amino acids is presented in parentheses.
Basic: arginine (R), lysine (K), histidine (H)
Acidic: glutamic acid (E), aspartic acid (D)
Polar: glutamine (Q), asparagine (B)
Hydrophobic: leucine (L), isoleucine (I), valine (V)
Aromatic: phenylalanine (F), tryptophan (W), tyrosine (Y)
Small: glycine (G), alanine (A), serine (S), threonine (T), methionine (M)
Amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992.
Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).
Any reference sequence described herein may differ from any other reference (e.g. wild-type) sequence due to natural sequence variation, yet be substantially homologous (e.g. have high sequence identity). The skilled person also understands how to employ appropriate sequence alignment to identify homologous/analogous nucleic acid/polypeptide positions amongst such sequences.
Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position—Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al., Align-M—A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004).
Thus, percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the “blosum 62” scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes).
The percent identity is then calculated as:
Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled person with a general dictionary of many of the terms used in this disclosure.
This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
The headings provided herein are not limitations of the various aspects or embodiments of this disclosure.
Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation. The term “protein”, as used herein, includes proteins, polypeptides, and peptides. As used herein, the term “amino acid sequence” is synonymous with the term “polypeptide” and/or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. In some instances, the term “amino acid sequence” is synonymous with the term “enzyme”. The terms “protein” and “polypeptide” are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues may be used. The 3-letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.
Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be defined only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.
It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a mutation” includes a plurality of such polymorphisms and reference to “the mutation” includes reference to one or more polymorphisms and equivalents thereof known to those skilled in the art, and so forth.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.
Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.
The samples tested and validated for resistance were obtained from Ireland, London, the midlands, north, south and east of England, Scotland and Wales. Thus, the results provided are representative of HSV strains present over widespread geographic regions. Samples were isolated from patients infected with a HSV and who did not respond (e.g. respond sufficiently) to treatment with one or more antiviral drugs (e.g. acyclovir, penciclovir, foscarnet, cidofovir).
The viruses were typically isolated from clinical swabs taken from patient lesions. The viruses were either cultured directly from the patient samples, or the virus or relevant segments thereof were amplified and cloned into recombinant vectors (e.g. using unique restriction sites or by homologous recombination). The recombinant vectors are then typically introduced into cells by transfection to produce recombinant and/or pseudotyped viruses.
Isolated viruses are typically titrated e.g. using monolayers of African green monkey kidney cells (Vero cells).
The samples were anonymized e.g. by removal of any patient identifiable information and assignment of a non-specific project number.
Mutations were typically detected by DNA sequencing.
To prepare viral DNA for sequencing, confluent monolayers of cells (e.g. Vero cells) were infected at 5 PFUs/cell for 24 to 48 hours, until cytopathic effect became apparent. Viral supernatants were harvested by freeze-thawing and passed through a 0.45 μM filter. Four ml of the virus supernatant was incubated with 20 U/ml DNase (Promega) for 3 hours at 37° C. before loading onto a 1.5 ml 20% sucrose cushion and centrifuged at 100,000 g for 1 hour. Viral pellets were re-suspended in TE containing DNase chelator and extracted with the Pure-Link Viral RNA/DNA Mini Kit (Invitrogen) following manufacturer's instructions. Alternatively, viral DNA was extracted directly from a clinical sample using QIAamp DNA mini kit (Qiagen) or Pure-Link RNA/DNA mini kit (Invitrogen).
Regions of interest (e.g. the UL23 and/or UL30 genes) were then amplified by PCR. Primers which flank the UL23 and/or UL30 genes were typically used, although primers designed to amplify only a fragment of said gene in which a polymorphism may be expected to occur may also be suitable. The resulting amplicons were then sequenced using typical DNA sequencing technology available to the skilled person. The DNA sequences are then used to determine the corresponding polypeptide sequence.
The nucleotide and/or amino acid changes in the amplified sequence are detected by comparison with wild-type reference strains (or pretreatment sequences). Suitable wild-type reference strains include GenBank accession number JN555585.1 and JQ673480.1 as reference for HSV-1; and Z86099.2 and JN561323.2 as reference for HSV-2.
Viral sample were typically phenotypically characterised using the plaque reduction assay.
Viral isolates were used to infect a sub-confluent monolayer of Vero cells at a concentration of 75 or 50 plaque forming units (PFU) per well for HSV-1 and HSV-2, respectively. After 1 hour incubation at 37° C. cells were overlaid with CMC medium (4% Carboxymethyl cellulose in PBS) containing a serial dilution of the antiviral drugs acyclovir (ACV), pencyclovir (PCV), cidofovir (CDV) and foscarnet (FOS) or CMC alone, as a no drug control, and incubated for a further 72 hours until plaques became apparent. CMC medium typically used to create a semisolid interface and prevents indiscrimate viral spreading. Other materials (e.g. agar) may also be used to create this interface.
Cells were fixed with 10% formalin and stained with crystal violet before enumeration of the plaques (microscopic observation or use of fluorescent antibodies for detection is also suitable). The data obtained from these experiments were then expressed as percent inhibition of viral infectivity relative to the no drug control. The data was then used to determine IC50 values for all four drugs using linear regression—i.e. dose-response curves were constructed from which the drug concentrations required to inhibit virus replication by 50% (IC50) were determined.
Definitions of phenotypic drug susceptibility classification as sensitive or resistant were as follows: ACV, <3 μM or >40 μM; PCV, <10 μM or >40 μM; CDV, <24 μM or >30 μM; FOS, <250 μM or >400 μM; and ACV, <6.5 μM or >40 μM; PCV, <38 μM or >40 μM; FOS, <250 μM or >400 μM for HSV-1 and HSV-2, respectively. Any IC50 values falling in between these sensitive and resistant cut-offs were reported as intermediate resistance.
The ratio of the IC50 of patient-derived virus may also be divided by that of a wild-type reference virus (e.g. a virus known to be susceptible to the drug under investigation) to provide a fold change and determine the susceptibility of the patient-derived virus. A fold change greater than one means that the patient-derived virus is less susceptible to that particular drug compared with wild-type virus and vice versa.
A fold change greater than one does not necessarily mean that the patient will not respond to the treatment; therefore, IC50 or fold change cutoff values have to be determined for each drug at which a patient-derived virus is considered to be susceptible or resistant. Different types of cutoff values can be used but the most pertinent is the ‘clinical cutoff’ which takes into consideration the relationship between IC50 or fold change values and virological response or clinical outcome.
Viral samples were isolated from patients suspected of being infected with an antiviral-drug resistant HSV. The mutation (where relevant) resulting in resistance was detected by DNA sequencing as described above. Aligning the TK and/or DNA pol sequence of the antiviral-resistant virus with that of a wild-type reference sequence demonstrated the relevant mutation.
The phenotype (i.e. reactivity to a drug) of the virus was characterised by the plaque reduction assay as described above. The present inventors have characterised the resistance-causing phenotype of a large number of TK and DNA pol mutations. Numerous natural polymorphisms (which do not cause drug resistance) have also been detected and characterised. The mutations, polymorphisms and associated phenotypes are demonstrated in Tables 4-7.
Definitions of phenotypic drug susceptibility classification: Acyclovir (ACV): <3 uM, 3-40 uM, >40 uM; Pencyclovir (PCV): <10 uM, 10-40 uM, >40 uM; Foscarnet (FOS): <250 uM, 250-400 uM, >400 uM; Cidofovir (CDV): <24 uM, 24-30 uM, >30 uM for HSV-1 and ACV: <6.5 μM, 6.5-40 μM, >40 μM; PCV: <38 μM, 38-40 μM, >40 μM; FOS: <250 μM, 250-400 μM, >400 μM for HSV-2, for sensitive, intermediate and resistant samples, respectively.
Table 4 presents ‘natural’ polymorphisms and resistance-associated substitutions and indels in HSV-1 TK gene and their effect on antiviral susceptibility. R=resistance (ACV and PCV IC50>40 μM; FOS IC50>400 μM; CDV IC50>30 μM); IR=intermediate resistance (ACV IC50>3 μM<40 μM; PCV IC50>10 μM<40 μM; FOS IC50>250<400 μM; CDV IC50>24 μM<30 μM); S=susceptible (ACV IC50<3 μM; PCV IC50<10 μM; FOS IC50<250 μM; CDV IC50<24 μM); ND=not done; amutation in non-conserved region in a sample which also contains Y239H in TK gene at a position associated with resistance; bsample also contains S914L and A1169AT mutations in non-conserved region of DNA pol gene; % ample also contains A1169AT mutations in non-conserved region of DNA pol gene; dsample also contains known resistance-associated mutation T287M in TK gene.
Table 5 presents ‘natural’ polymorphisms and resistance-associated substitutions and indels in HSV-2 TK gene and their effect on antiviral susceptibility. R=resistance (ACV and PCV IC50>40 μM; FOS IC50>400 μM); IR=intermediate resistance (ACV IC50>6.5 μM<40 μM; PCV IC50>38 μM<40 μM; FOS IC50>250<400 μM); S=susceptible (ACV IC50<6.5 μM; PCV IC50<38 μM; FOS IC50<250 μM); ND=not done; bmutation in non-conserved region of TK gene but virus was not isolated and therefore phenotypic drug susceptibility testing could not be performed
Table 6 presents ‘natural’ resistance-associated substitutions and indels in HSV-1 pol gene and their effect on antiviral susceptibility. R=resistance (ACV and PCV IC50>40 μM; FOS IC50>400 μM; CDV IC50>30 μM); IR=intermediate resistance (ACV IC50>3 μM<40 μM; PCV IC50>10 μM<40 μM; FOS IC50>250<400 μM; CDV IC50>24 μM<30 μM); S=susceptible (ACV IC50<3 μM; PCV IC50<10 μM; FOS IC50<250 μM; CDV IC50<24 μM). Also contain A1169T mutation in non-conserved region; amutation in non-conserved region in a sample which also contains the deletion (C) nt 881-885 in TK gene associated with resistance; bmutation in non-conserved region in a sample which also contains the mutation M1211 in TK gene; cmutation in non-conserved region in a sample which also contains the mutation R176stop in TK gene associated with resistance; amutation in non-conserved region in a sample which also contains the mutation Q261stop in TK gene associated with resistance; amutation in non-conserved region in a sample which also contains the deletion (G) nt 430-436 in TK gene associated with resistance; fmutation in non-conserved region in a sample which also contains the mutation S724N in DNA pol gene associated with resistance; gmutation in non-conserved region in a sample which also contains the mutation L49R in TK gene; imutation in non-conserved region in a sample which also contains the deletion (C) nt 548-553 in TK gene associated with resistance; jmutation in non-conserved region in a sample which also contains deletion (G) nt 430-436 in TK gene associated with resistance; kmutation in non-conserved region in a sample which also contains deletion (G) nt 430-436 in TK gene associated with resistance; lmutations in conserved region (and for L802R on site associated with resistance) in a sample which also contains mutation R176W in TK gene associated with resistance; mmutation in conserved region in a sample which also contains deletion (G) nt 430-436 in TK gene associated with resistance; nmutation in non-conserved region in a sample which also contains mutation R216C in TK gene associated with resistance; omutation in non-conserved region in a sample which also contains mutation deletion (C) nt 896-900 in TK gene associated with resistance; p mutation in non-conserved region in a sample which also contains mutation V204G in TK gene associated with resistance; qmutation in non-conserved region in a sample which also contains mutation A93V in TK gene associated with resistance; rmutation in non-conserved region in a sample which also contains mutation insert (G) nt 430-436 in TK gene associated with resistance; smutation in non-conserved region in a sample which also contains mutation T287M in TK gene associated with resistance; tmutation in non-conserved region in a sample which also contains mutation deletion (C) nt 881-885 in TK gene, also Q substitution at this site is a known polymorphism
Table 7 presents resistance-associated substitutions in HSV-2 pol gene and their effect on antiviral susceptibility. R=resistance (ACV and PCV IC50>40 μM; FOS IC50>400 μM); IR=intermediate resistance (ACV IC50>6.5 μM<40 μM; PCV IC50>38 μM<40 μM; FOS IC50>250<400 μM); S=susceptible (ACV IC50<6.5 μM; PCV IC50<38 μM; FOS IC50<250 μM); amutation in non-conserved region in a sample which also contains mutation S729N in DNA pol associated with resistance; bmutation in non-conserved region in a sample which also contains mutation Q90stop in TK gene; cmutation in non-conserved region in a sample which also contains mutation deletion (G) nt 276-280 in TK gene.
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1809135.5 | Jun 2018 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2019/051546 | 6/4/2019 | WO | 00 |
